| Animal fur skin color is determined by the content and type of melanin in the hair follicle,the colors of the fur coat is the main raw material of today textile industry.Melanin is mainly formed in melanocytes The survival and development of melanocytes and the synthesis of pigment are closely related to the expression of MITF.MITF can regulate enzymes related to pigmentation,such as TYR,TYRP1 and TYRP2,which are important downstream genes of MITF and also key enzymes in the pigment synthesis process.Therefore,many different external signals affect melanin production by regulating the expression of MITF and its downstream genes.MicroRNA(miRNA)is a highly conserved non-coding RNA.miRNAs inhibit target genes by binding to target genes.In this study,TargetScan software was used to predict that miR-101-3p and miR-144-3p have the same binding sites in mitf 3'UTR.The function of miR-101-3p and miR-144-3p in melanocyte was detected by over-expression of miR-101-3p and miR-144-3p in different ratio group.Real-time PCR,western blotting and immunohistochemistry was used to detect the function and further confirm the regulation mechanism of miR-101-3p and miR-144-3p1.The homology of miR-101-3p and miR-144-3p in human,mouse,cow,chicken and other species is extremely high at about 100%.The homology of Mitf 3'UTR in humans,mice,chickens,sheep and other species is extremely high almost above 80%.miR-101-3p and miR-144-3p have the same binding sites in Mitf 3'UTR.2.The endogenous expression of miR-101-3p,miR-144-3p and Mitf,Tyr,Tyrp1 and Tyrp2 in alpaca melanocytes were detected by PCR.The result show that miR-101-3p,miR-144-3p and Mitf,Tyr,Tyrp1 and Tyrp2 all express in alpaca melanocytes3.The over expression of miRNA by transfecting miRNA mimics in melanocytes and inhibition of miRNA by transfecting miRNA inhibitor.The result show that the melanin decrease in miR-144-3p mimics group(P<0.001),miR-101-3p mimics group(P<0.001)and miR-144-3p and miR-101-3p mimics(1:1)group(P<0.001).The amount of melanin produced in the co-transfection of miR-101-3p mimics and miR-1 44 mimics cells was significantly higher than that transfected with miR-144-3p mimics(P<0.05),but lower than that of melanocytes transfected with miR-101-3p mimics(P<0.05).The levels of melanin in melanocytes transfected miR-101-3p mimics and miR-144-3p were significantly higher than normal controls(P<0.01).The levels of melanin in melanocytes transfected miR-101-3p mimics and miR-144-3p were significantly lower than normal controls after transfected with miR-101-3p inhibitors and miR-144-3p inhibitors(P<0.05).The production of melanin in miR-101-3p&miR-144-3p(1:1)inhibitors was significantly higher than that in transfected miR-144-3p inhibitors melanocytes(P<0.05),lower than those transfected with miR-101-3p inhobitors(P<0.01).4.The expression of miR-101-3p,miR-144-3p,Mitf,Tyr,Tyrp1 and Tyrp2 in transfected melanocytes was detected by qRT-PCR.The protein expression was detected via western blotting and immunohistochemistry.The results show that there is no significant change in the mRNA level of Mitf.The mRNA expression of Tyr,Tyrp1 and Tyrp2 in miR-101-3p mimics and miR-144-3p mimics co-transfection groups is higher than that in miR-144-3p mimics transfection group(P<0.05),lower than the miR-101-3p mimics transfection group(P<0.05).The results of Western blotting and immunohistochemistry show that the protein expression of MITF,TYR,TYRP1 and TYRP2 are significantly decreased after transfecting miRNA mimics comparing with normal group(P<0.001).The protein expression of TYR,TYRP1 and TYRP2 in miR-101-3p&miR-144-3p(1:1)mimics co-transfection group was higher than miR-144-3p mimics transfection group(P<0.05),lower than miR-101-3p mimics transfection group(P<0.05).The effect of miR-101-3p and miR-144-3p in melanocyte was studied at the cellular level used miR-101-3p transfection group,miR-144-3p transfection group,and co-transfection group.The results show that miR-101-3p and miR-144-3p could indirectly regulate the expression of TYR,TYRP1,TYRP2 by regulating the expression of MITF,thereby affecting the synthesis of melanin.At the same time,it was also revealed that miRNAs that have the same binding site in the same binding site act independently and do not compete with each other or antagonize with each other.The results not only enrich the theoretical knowledge of melanin formation mechanism,but also reveal the function mechanism of miRNAs with the same binding site in the same target gene for the first time.Providing important genetic resources and experimental basis for genetic engineering and genetic modification of miRNAs. |
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